ES2799581T3 - Assay to capture and detect circulating multiple myeloma cells in the blood - Google Patents

Abstract

A method for capturing, isolating and analyzing circulating multiple myeloma cells in a blood sample obtained from a test subject comprising (a) contacting said sample with colloidal magnetic particles that are conjugated to a first ligand, which is anti- CD138, and a second ligand, which is anti-CD38; (b) subjecting the sample from step (a) to a magnetic field to produce a separate faction of circulating multiple myeloma cells attached to magnetic particles; (c) treating the sample from step (b) with an additional first marker; and (d) analyzing circulating multiple myeloma cells.

Description

DESCRIPTION

Assay to capture and detect circulating multiple myeloma cells in the blood

Related requests

This non-provisional filing claims priority to a provisional patent application, United States Patent Application Serial No. 61 / 510,170, filed on July 21, 2011.

Background of the invention

Multiple myeloma (also known as myeloma or plasma cell myeloma) is a progressive hematologic cancer of plasma cells. The condition is characterized by an excessive number of plasma cells in the bone marrow and the overproduction of intact monoclonal immunoglobulin or free monoclonal light chains.

Clinically, the disease is diagnosed, classified, and treated based on a variety of parameters including the mass of myeloma tumor cells based on the amount of monoclonal protein (or myeloma) (M protein) in serum and / or urine, together with serum hemoglobin and calcium concentrations, the number of lytic bone lesions based on a skeletal study, and the presence or absence of renal failure. Additional approaches to characterizing the condition include the detection of greater than ten percent (10%) of plasma cells in a bone marrow examination, the presence of soft tissue plasmacytomas, and the detection of the serum kappa and lambda immunoglobulin light chain. free. Examination of the bone marrow is performed using standard histology and immunohistochemical techniques. Additional cytogenetics of bone marrow samples can be performed to determine prognosis. Follow-up surveillance consists of chemical and bone marrow evaluations, if clinically indicated due to its invasive nature.

Currently, bone marrow flow cytometric analysis is being evaluated as a tool for characterization of disease and to distinguish between neoplastic plasma cell disorders from normal plasma cells, and to detect minimal residual disease. However, this approach continues to rely on an invasive procedure. There is a significant need to develop less invasive techniques to detect, monitor, and characterize disease throughout its history, and the presence of these cells in the blood can provide that opportunity.

Additionally, more sensitive tools need to be developed for more accurate risk assessment and monitoring of disease progression in earlier stages of the disease, including monoclonal gammopathy of undetermined significance (MGUS) and latent multiple myeloma. Some research data suggests that circulating plasma cells can be detected in the early stages of the disease and can be correlated with prognosis, supporting the use of a standardized methodology to capture, enumerate, and characterize these cells in the early stages of the disease.

The general consensus within the bibliography (Report of the European Myeloma NetWork on Multiparametric Flow Cytometry in Multiple Myeloma and Related Disorders. Andy C. Rawstron et al. Haematologica, 2008; 93 (3)) for the identification of abnormal plasma cells, particularly By flow cytometry, he has included several key biomarkers consisting primarily of CD138, CD38, and CD45. Additional biomarkers such as CD19 and CD56 have also shown diagnostic utility.

The present invention investigates circulating myeloma cells to evaluate whether these particular biomarkers, either alone or in combination with one or more additional biomarkers or with FISH, can be used for both the capture and detection of abnormal circulating plasma cells, including detection of minimal residual disease. FISH can be used to detect numerous cytogenetic abnormalities that have been described in multiple myeloma. Translocations at the IGH locus, t (4; 14) and deletions at the p53, del (17p) locus have been shown to have prognostic value for event-free and overall survival in multiple myeloma. (Genetic Abnormalities and Survival in Multiple Myeloma: The Experience of the InterGroupe Francophone du Myelome. Herve Avet-Loiseau et al. Blood, 2007; 109: 3489-3495). These probes and various other multiple myeloma markers are available from the Poseidon catalog and could be adapted for use with the CellTracks® platform.

Immunomagnetic screening kits are commercially available that use CD138 magnetic particles. Stem Cell Technologies has an EasySep® human CD138 positive selection kit that can select CD138 positive cells from bone marrow and peripheral blood mononuclear cells (PBMC) and Miltenyi Biotech has the CD138 microbeads for selection of CD138 positive cells from bone marrow, PBMC and whole blood. Analysis of collected samples is typically performed using flow cytometry.

WO 99/41613 A1 relates to methods for the rapid and efficient isolation of circulating cancer cells.

Summary of the invention

The invention provides a method for capturing, isolating and analyzing circulating multiple myeloma cells in a blood sample obtained from a test subject comprising: (a) contacting said sample with colloidal magnetic particles that are conjugated to a first ligand, which is anti-CD138 and a second ligand, which is anti CD-38; (b) subjecting the sample from step (a) to a magnetic field to produce a separate faction of circulating multiple myeloma cells attached to magnetic particles; (c) treating the sample from step (b) with an additional first marker; and (d) analyzing circulating multiple myeloma cells.

The invention also provides a method for determining whether a patient is a likely candidate for therapeutic intervention for diseases associated with abnormal plasma cells comprising: (a) processing said patient's blood to determine how many CMMC are in the sample, wherein the processing comprises the method of the invention; and (b) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range.

The invention also provides a method for determining whether a patient undergoing therapeutic intervention is reducing the number of CMMC, or for determining whether a patient who had abnormal plasma cell disease and has been successfully treated for said disease remains in remission; the method comprising: (a) processing the blood of said patient to determine how many CMMC are in the sample at a first time point, wherein the processing comprises the method of the invention; (b) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range; (c) processing the blood of said patient to determine how many CMMC are in the sample at a second time point, wherein the processing comprises the method of the invention; and (d) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range; (e) compare the numbers in steps (b) and (d).

The invention also provides a reagent for capturing circulating multiple myeloma cells comprising colloidal magnetic particles and at least two ligands, wherein the at least two ligands comprise anti-CD-138 and anti-CD-38.

Disclosed herein is a method for the capture and detection of circulating plasma cells (CPC) and abnormal plasma cells or multiple myeloma cells ("CMMC") that includes detection of minimal residual peripheral blood disease. Disclosed herein is a non-invasive means of detecting very low amounts of CMMC in millimeter volumes of blood sample to detect, monitor, and characterize disease throughout its history, as well as providing the most sensitive tools for further evaluation. Accurate risk and monitoring for disease progression in the early stages of the disease including detection of circulating plasma cells in the early stages of the disease, including monoclonal gammopathy of undetermined significance (MGUS) and latent multiple myeloma. Capturing and characterizing these circulating peripheral blood plasma cells may provide new biomarkers for the management of multiple myeloma patients.

Blood is collected into CellSave tubes that contain a preservative that allows for the transport and storage of blood while minimizing cell degradation. Cells are captured using colloidal magnetic particles conjugated to Syndecan-1 or CD138, a cell surface marker present on plasma cells. Once captured, cells are labeled with the additional cell markers CD38-PE (Phycoerythrin), CD19 and CD45-APC (allophycocyanin) and CD56-FITC (fluorescein isothiocyanate) to differentiate multiple myeloma cells from contaminating background leukocytes. (white blood cells). Ferrofluidic and cell marker reagents are part of a new CellSearch® CMMC service kit. The kit consists of 7 components, 4 of which are identical to the reagents found in the Cellsearch® Epithelial Cell kit. These 4 common reagents are Capture Enhancement Reagent, Perm Reagent, Nucleic Acid Stain, and CellFix. The 3 new reagents consist of the CD138 ferrofluid, a staining reagent consisting of CD38-PE, CD19, and CD45-APC, and a separate marker staining reagent consisting of CD56-FITC.

Brief description of the figures

Figure 1 illustrates the reactivity of CD 138 antibodies with certain cell lines.

Figure 2 illustrates the reactivity of CD 38 antibodies with certain cell lines.

Figure 3 illustrates the reactivity of different CD19 APC antibody tests in PBMC.

Figure 4 illustrates CD 19 APC staining at various dilutions.

Figure 5 illustrates CD 56 antibody staining in PBMC.

Figure 6 illustrates CD 56 staining in various cell lines.

Figure 7 illustrates staining without CD56.

Figure 8 illustrates CD 56 FITC staining of a cell line at one dilution.

Figure 9 illustrates CD 56 FITC staining of a cell line at one dilution.

Figure 10 illustrates CD 56 FITC staining of a cell line at one dilution.

Figure 11 illustrates representative images of MM 1S cells.

Figure 12 illustrates representative images of H929 cells.

Figure 13 illustrates representative images of the leftover white blood cells.

Figure 14 illustrates images of a patient sample.

Figure 15 illustrates images of a patient sample.

Figure 16 illustrates images of a patient sample.

Figure 17 illustrates images of a patient sample.

Figure 18 illustrates images of normal donors.

Detailed description

The invention includes a method of capturing, isolating and analyzing circulating multiple myeloma cells in a blood sample obtained from a test subject comprising

(a) contacting said sample with colloidal magnetic particles that are conjugated to a first ligand, which is anti-CD138, and a second ligand, which is anti-CD38;

(b) subjecting the sample from step (a) to a magnetic field to produce a separate fraction of circulating multiple myeloma cells attached to magnetic particles

(c) treating the sample from step (b) with an additional first marker; and

(e) analyzing circulating multiple myeloma cells.

As used herein, the term "sample" refers to an amount of blood, preferably expressed as a volumetric measurement. The preferred volume of a blood sample is about 2 ml to 10 ml, more preferably 3-7.5 ml, most preferably 4 ml. The term "colloidal magnetic particles" refers to particles that are metallic or organometallic. Examples of such particles are disclosed in US Patent Nos. 5,597,531; 5,698,271; 5,698,271; 6,365,662, particularly for its description of such colloidal magnetic particles. They can optionally be coated with a polymer, preferably a polymer of biological origin such as bovine serum albumin and casein.

The term "ligand" refers to proteins that bind to CMMC cell-associated markers. Preferred proteins are antibodies. The ligands are anti-CD 138 and anti-CD 38. Such ligands can be conjugated to colloidal magnetic particles by methods that are substantially similar to the methods disclosed in 6,365,662. In step (a) of the invention two or more ligands can be used.

The term "magnetic field" can be produced by any of several methods, particularly by two magnetic separators substantially as described in US Patent No. 7,901,950. The term "additional marker" means a cell-associated protein that is specific for CMMC or excludes CMMC. Such proteins include, but are not limited to, antibodies selected from the group consisting of anti CD19 and anti CD45, anti CD 56, anti lambda, anti kappa, anti CD 200, anti Ki67. Such antibodies can be labeled with indicators such as phycoerythrin, fluorescein isothiocyanate and allophycocyanin, and it is preferable that they are labeled with one or more labels. The additional label can include nucleic acid dyes such as DAPI. The preferred additional marker is selected from the group consisting of anti CD19 and anti CD45. In step (c) of the invention two or more additional markers may be used and it is preferred that at least two additional markers are used, more preferably three additional markers, most preferably four additional markers.

The term "analyze" means to evaluate the magnetically captured sample to determine one or more of the following: whether the sample contains CMMC. Such identification can be done visually or electronically to determine the degree of fluorescence of a magnetically captured sample. Such methods of analysis are disclosed in US Patent No. 7,011,794. In particular, magnetically captured samples that are positive for CD38 and negative for CD19 and CD45 are identified as CMMC.

The invention includes a method of determining whether a patient is a likely candidate for therapeutic intervention for diseases associated with abnormal plasma cells comprising

(a) processing the blood of said patient to determine how many CMMC are in the sample, wherein the processing comprises the method of the invention; and

(b) determining by counting whether the number of CMMC cells present in said sample is equal to or greater than or equal to the normal range.

As used herein, the term "sample" has the aforementioned meaning and the preferred range. The term "processing" means treating a patient's blood sample by the methods described herein to isolate and identify CMMC.

The term "therapeutic intervention" means to seek or obtain any medical intervention to treat diseases associated with abnormal plasma levels. Such diseases include, but are not limited to, multiple myeloma, MGUS, and smoldering multiple myeloma. Such therapeutic intervention includes, but is not limited to, visiting a physician, obtaining therapeutic treatment such as radiation, and treatment with drugs that treat any of the diseases associated with abnormal plasma levels, and monitoring the effect of said therapeutic treatments. For example, if a patient is being treated with a drug, the patient's CMMC levels can be assessed during the course of treatment to determine if the drug is working. Such drugs include, but are not limited to, dexamethasone, cyclophosphamide, vincristine, bortezomib, melphalan, zometa, aloxy, lenalidomide, doxirubicin, and the like.

The term "normal range" means the number of CMMC cells present in a sample population that do not have diseases associated with abnormal plasma cells. Preferably, the normal range is less than 7 CMMC in a blood sample from about 2 ml to about 10 ml. Preferably, the normal range is less than 7 CMMC in a blood sample from about 3 ml to about 7.5 ml. The term "greater than normal range" is a number of CMMC that exceeds the normal range. The higher this number, the more likely it is that the patient has one of the diseases associated with abnormal plasma cells. If a patient has between 8 and 20 CMMC in a blood sample, that patient has a higher chance of having one of the diseases associated with abnormal plasma cells. If a patient has between 21 and 49 CMMC the patient has an elevated level and is more likely to have one of the diseases associated with abnormal plasma cells, without a patient has between 50 and tens of thousands of CMMC that patient has a highly elevated level and even more likely to have one of these diseases.

Furthermore, the invention includes a method for determining whether a patient undergoing therapeutic intervention is reducing the number of CMMC, or for determining whether a patient who had an abnormal plasma cell disease and has been successfully treated for said disease, still in remission; the method comprising

(a) processing the blood of said patient to determine how many CMMC are in the sample at a first point in time, wherein the processing comprises the method of the invention;

(b) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range;

(c) processing the blood of said patient to determine how many CMMC are in the sample at a second point in time, wherein the processing comprises the method of the invention;

(d) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range; and

(e) compare the numbers in steps (b) and (d).

All the terms defined above have the same meaning and preferred range.

Furthermore, the invention includes a reagent for capturing circulating multiple myeloma cells comprising colloidal magnetic particles and at least two ligands, wherein the at least two ligands they comprise anti-CD138 and anti-CD38.

All terms defined above have the same meaning and preferred ranges.

Circulating multiple myeloma cells (CMMC), a form of abnormal plasma cells, captured from the blood, have been captured and analyzed using the CellTracks® AutoPrep® and CellTracks Analyzer II® systems. In this procedure, a combination of capture reagent (ferrofluid) and fluorescent biomarkers (such as anti-CD38-Phycoerythrin (PE) antibody) and dyes (such as DAPI nucleic acid stain) are used to identify abnormal plasma cells and distinguish them from leukocytes and polluting wastes. CD138 or Syndecan-1 is a cell surface marker found in mature plasma cells and in malignant plasma cell neoplasms such as multiple myeloma but not in other normal peripheral blood leukocytes. For this reason, anti-CD138 was coupled to ferrofluid magnetic nanoparticles, which are used to magnetically select circulating plasma cells from a peripheral blood sample. Various fluorescent biomarkers are used to detect abnormal plasma cells from contaminating leukocytes. Anti-CD38 is conjugated to phycoerythrin (PE) and is used as a positive marker for the detection of plasma cells. However, since CD38 is also found in some types of leukocytes (activated T and B cells), the assay also uses allophycocyanin-conjugated anti-CD45 (APC) and allophycocyanin-conjugated anti-CD19 (APC) as a negative marker. CD45 is a pan-leukocyte marker found in peripheral blood leukocytes, and CD19 is a B-cell specific marker. Myeloma cells are functionally differentiated B cells that express neither CD45 nor CD19. A final marker in this assay is anti-CD56 conjugated to fluorescein isothiocyanate (FITC). CD56 can be found in some peripheral leukocyte subsets such as NK cells, but it is also expressed in 75% of myeloma cells and is often associated with a worse patient prognosis. Therefore, although CD56 is neither a positive nor a negative marker for multiple myeloma, its expression levels in cells can be controlled during drug therapy of the patient.

The assay was initially developed using cell lines such as RPMI 8226, H929, and MM.1S to evaluate different antibodies against markers that were determined to be present on multiple myeloma cells including CD138, CD38, and CD56. Since these cell lines were negative for CD45 and CD19, PBMC were used instead to test for those antibodies.

The enriched and stained cells were transferred to a CellTracks® and MagNest® cartridge for magnetic mounting. The cartridge was scanned using CellTracks Analyzer II®. Individual cell images were presented to the operator for review, and scored as CMMC, based on fluorescence and cell morphology. In a model spike system, the assay consistently recovered ~ 60% of the H929 multiple myeloma cell line cells added to 4.0 ml of blood from healthy donors. The assay was linear in the range tested from 0 to 2000 H929 cells with spikes (r2 0.98, slope 0.50, intercept 10). The assay was validated using blood from healthy age-compatible donors (n = 22) and patients with multiple myeloma (n = 66) and MGUS (n = 7). In 4.0 ml of blood from normal donors, 0 CPCs were detected in 12/22 (55%) and low numbers (1-6 CPCs) were detected in 10/22 (45%) of the samples. Interestingly, a CD56 positive CPC was found in a normal donor. CMMC in multiple myeloma patients ranged from 0 to 17,000 / 4.0ml of blood. One or more CMMC were detected in 91% of patients,> 5 in 68%,> 10 in 58%, and> 100 in 35%. The expression of CD56 was highly variable in the patient population. CMMC in patients with MGUS ranged from 0 112 / 4.0 ml of blood. One or more CMMC were detected in 6/7 of the patients,> 5 in 4/6,> 10 in 2/6 and> 100 in 1/6.

To further characterize CMMC and differentiate CPC from CMMC, a fluorescent in situ interphase hybridization (FISH) assay was developed for use with the capture and detection system described above. A four-color FISH probe was used to simultaneously detect high-risk mutations. The following examples illustrate the invention.

Examples

Abbreviations

PE-Phycoerythrin

FITC-fluorescein isothiocyanate

APC-Allophycocyanin

PBMC-peripheral blood mononuclear cells

Antibody sources

CD138:

Gen-Probe Diaclone SAS

1 Bd A Fleming, BP 1985

F-25020 Besancon Cedex, France

CD38 and CD19

R&D Systems

614 McKinley Place NE

Minneapolis, MN 55413

Example 1 Capture targets

Figure 1 shows that of the anti-CD138 antibodies tested for reactivity with RPMI 8226, H929 and MM.1S cell lines, the best performing antibody was clone B-A38. The cell lines were first labeled with the different antibodies, which were all mouse anti-human antibodies, then subsequently labeled with an anti-mouse PE conjugate and analyzed by flow cytometry. Clone B-A38 provided the highest fluorescent staining in all multiple myeloma cell lines.

Example 2 Detection targets

Figure 2 shows that of the anti-CD38 antibodies tested for reactivity with RPMI 8226, H929 and MM.1S cell lines, the best performing antibody was clone 240742. Cell lines were initially tested using an anti-CD38 conjugate. -FITC direct but later it was discovered that the FITC conjugate was not adequate enough for detection on the CellTracks® platform. A PE conjugate of this antibody was subsequently prepared and tested and found to be suitable for detection.

Example 3 Determination of dilution

Anti CD19 and anti-CD45, both as APC conjugates, were chosen as negative detection markers since the absence of both is indicative of abnormal plasma cells. Anti-CD45APC is already a component of the CellSearch® CTC staining reagent, so no further optimization was necessary. And since none of the myeloma cell lines under evaluation expressed CD19, PBMCs were used to evaluate the different anti-CD19APC clones. The results of the anti-CD19 tests in PBMC can be seen in Figure 3. Staining was performed according to the manufacturer's recommended protocols on PBMC collected from EDTA and CellSave tubes. Clone SJ25C1 was chosen as the best performing conjugate. The conjugate was then tested at various dilutions in the same reaction volumes used in AutoPrep®, Figure 4. A dilution of 1: 5 was chosen as the final dilution for staining.

Example 4 CD56 and PBMC staining

Anti-CD56 was chosen as the FITC marker reagent, since it is expressed in 75% of cases of myeloma with abnormal expression. The tests were carried out on cell lines (Figure 6) and PBMC (Figure 5) according to the protocol recommended by the manufacturer. NCAM 16.2 was chosen as the best performing conjugate.

Example Dilutions of 5 CD 56 FITC

The anti-CD56 FITC NCAM 16.2 conjugate was tested at various dilutions with H929 cells in AutoPrep®, see Figure 7-10. No clear dilution tested was better in the cell line. A dilution of 1: 4 was chosen as the final dilution for staining until patient samples could be tested to help determine the optimal concentration.

Example 6 Images

A CMMC prototype kit was then constructed consisting of anti-CD138 ferrofluid, staining reagent consisting of anti-CD38 PE, anti-CD45 APC and anti-CD19 APC, and an anti-CD56 FITC marker reagent. The remaining components of the kit were Capture Enhancement Reagent (PN 7037), Permeabilization Reagent (PN 7038), Nucleic Acid Dye (PN 7041), and CellFix (PN 7042). The first round of testing used anti-CD138 ferrofluid at different concentrations. The final concentration of anti-CD38 PE was adjusted to 1 pg / ml (staining reagent concentration of 5.7 pg / ml) based on the above flow data. The final concentration of anti-CD45 APC was approximately 2 pg / ml (staining reagent concentration of 13 pg / ml, the same as in the CellSearch® CTC Kit) and the original anti-CD19 APC conjugate was diluted 1: 5 in the staining reagent. Anti-CD56 FITC was used at a concentration of 1: 4 in the marker reagent vial. H929 cells were added to 7.5 ml of CellSave blood and processed in AutoPrep® at ferrofluid concentrations of 135, 185, 220 and 270 pg / ml. The samples were then analyzed on the CellTracks Analyzer II® and the recovery of H929 cells reached a plateau of 55-60% at approximately 220 pg / ml.

Therefore, it was decided that the final concentration of ferrofluid in the kit would be 220 pg / ml per 7.5 ml of blood sample, which is similar to the concentrations used in many other CellSearch® kits.

CellTracks® images of recovered H929 and MM.1S cells can be seen in Figures 11 and 12 respectively. Take into account the higher CD56 staining of H929 cells compared to MM.1S cells. In Figure 13 you can see the excess white blood cells (CD38 / CD45 +).

Example 7 Patient samples

A total of 66 samples from multiple myeloma patients were analyzed for CMMC. These samples were obtained from Conversant and originally 7.5 ml of blood was analyzed using the CellTracks® CMMC Service Kit, but as it became apparent that many samples had a high number of CMMC, the decision was made to reduce the blood volume tested at 4 ml. The samples were processed in CellTracks® AutoPrep® and then scanned in CellTracks Analyzer II®. Figure 10 is a table of data generated from patient blood samples. CMMC in multiple myeloma patients ranged from 0 to 17,000 / 4.0ml of blood. One or more CMMC were detected in 91% of patients,> 5 in 68%,> 10 in 58%, and> 100 in 35%. The expression of CD56 was highly variable in the patient population. CMMC in patients with MGUS ranged from 0-112 / 4.0ml of blood. One or more CMMC were detected in 6/7 of the patients,> 5 in 4/6,> 10 in 2/6 and> 100 in 1/6. Figures 14-17 show some representative CellTracks® images of patient samples.

Legend of Table 1

Stage: a diagnostic classification of the extent and characteristics of myeloma cells comprising Stages I, II, and III. The number of cancer cells progresses from relatively few to moderate to relatively many as the Stage progresses. Additional symptoms such as M protein, anemia, and serum calcium also increase as the Stage progresses.

Treatment Status - Reflects disease status and treatment focus. Type of treatment: drug therapy or radiation therapy.

Volume: Volume of peripheral blood processed in the CellSearch® system.

Total Events - Total number of CellTracks® browser images presented to the user for manual multiple myeloma cell sorting.

Total MM Cells (CD38 +, CD19 / 45-) - Total number of images from total events that the user has determined meet the criteria for a multiple myeloma (MM) cell. These cells are CD38 positive and CD19 / 45 negative.

CD38 +, CD19 / 45-, CD56 +: the number of multiple myeloma cells that were positive for CD56.

CD38 +, CD19 / 45-, CD56-: the number of multiple myeloma cells that were negative for CD56.

Unassigned Events - Total events minus total MM cells, which were events that the user classified as multiple myeloma cells. Unassigned events are a combination of white blood cells, computer noise, or other debris not classified as MM cells.

Example 8 Normal subjects

Twenty-two normals of the same age were also tested. These samples were also obtained from Conversant and 4 ml of blood was analyzed with the CellTracks® CMMC Service Kit. Samples were processed in CellTracks® AutoPrep® and then scanned in CellTracks Analyzer II®. Table 2 is a table of data generated from the 4 ml blood samples. In 4.0 ml of blood from normal donors, 0 CPCs were detected in 12/22 (55%) and low numbers (1-6 CPCs) were detected in 10/22 (45%) of the samples. Interestingly, a CD56 positive CPC was found in a normal donor. See Figure 18. The average number of total browser events was approximately 1500.

To further characterize CMMC and differentiate CPC from CMMC, a fluorescent in situ inter-phase hybridization (FISH) assay was developed for use with the capture and detection system described above. A four-color FISH probe was used to simultaneously detect high-risk mutations, including two recurrent translocations of the IgH locus (t (4; 14) (p16; q32) and t (14; 16) (q32; q23)), as well as the deletion of the TP53 locus (A17p13). The FISH assay was verified in cell lines H929, M ^m 1 and U266, which showed mutations in t (4; 14), t (14; 16) and A17p13, respectively. The FISH assay was tested on 9 samples from CMMC patients and 8 samples provided evaluable results. Two samples showed t (4; 14) fusions, 3 patients showed aberrant FISH signal patterns indicating aneuploidy of chromosome 4 or 14, and the remaining patients showed normal FISH patterns.

Table 2: Table of CMMC from normal blood donors of the same age of 4 ml

Example 9 Capture using Anti-CD 38 and Anti CD 138 Patient samples

Anti CD138, the cell surface marker conjugated to used colloidal magnetic particles to capture myeloma cells in the present invention can be shed from the myeloma cell surface over time. CD38, a surface marker also present on myeloma cells, is not shed from the cell surface. A magnetic nanoparticle was developed that bound both anti-CD138 and anti-CD38 and tested with normal patient and donor samples for its ability to capture myeloma cells. Anti-CD38 and anti-CD 138, both conjugated with phycoerythrin (PE), and both recognizing an epitope different from the anti-CD38 and anti-CD 138 used in the manufacture of the magnetic nanoparticle, were used for detection together with anti-CD45 and anti-CD19 conjugated to allophycocyanin (APC).

A total of 22 samples from multiple myeloma patients were tested for CMMC using the alternative capture reagent. These samples were obtained from Conversant and 4 ml were run on CellTracks® AutoPrep® and then scanned on CellTracks Analyzer II®. Table 3 is a table generated from the patient samples. CMMC in multiple myeloma patients ranged from 0-2244 / 4.0 ml of blood. One or more CMMC were detected in 82% of patients,> 5 in 64%,> 10 in 59%, and> 100 in 23%.

Example 9 Capture using normal samples of Anti-CD 38 and Anti-CD 138

Twelve normal donors were also tested, using the same kit configuration as in Example 8. These samples were made from internal donors and 4 ml of blood was run on CellTracks® AutoPrep® and then scanned on the CellTracks Analyzer II®. Table 4 is a table generated from the 4 ml blood samples. In 4.0 ml of blood from normal donors, 0 CPCs were detected in 5/12 (42%) and low numbers (1-3 CPCs) were detected in 7/12 (58%) of the samples.

Table 4 of CPCs from normal donors

Claims (13)

1. A method of capturing, isolating and analyzing circulating multiple myeloma cells in a blood sample obtained from a test subject comprising (a) contacting said sample with colloidal magnetic particles that are conjugated to a first ligand, which is anti-CD138, and a second ligand, which is anti-CD38; (b) subjecting the sample from step (a) to a magnetic field to produce a separate faction of circulating multiple myeloma cells attached to magnetic particles; (c) treating the sample from step (b) with an additional first marker; and (d) analyzing circulating multiple myeloma cells. The method of claim 1, wherein the sample has a volume of from about 2 ml to about 10 ml, from about 3 ml to about 7.5 ml, or from about 4 ml. 3. The method of claim 1, wherein said colloidal magnetic particles are conjugated to more than two ligands, optionally wherein one of the ligands is anti-CD56. The method of claim 1, wherein said additional first marker is selected from the group consisting of DAPI, anti-CD38, anti-CD19 and anti-CD45 anti-CD138, anti-CD56, anti-lambda, anti-kappa anti-CD200, anti-Ki67. The method of claim 1, further comprising contacting the sample from step (b) with a second additional marker, optionally (i) wherein said second additional marker is selected from the group consisting of anti-CD19, anti -CD45 and DAPI. The method of claim 5 further comprising contacting the sample from step (b) with an additional third marker, optionally wherein said additional third marker is selected from the group consisting of anti-CD19, anti-CD45, and DAPI. . The method of claim 6 further comprising contacting the sample from step (b) with an additional fourth marker, optionally wherein the additional fourth marker is anti-CD45. The method of claim 1, further comprising contacting the sample from step (b) with four or more additional markers. 9. A method of determining whether a patient is a likely candidate for a therapeutic intervention for diseases associated with abnormal plasma cells comprising (a) processing the blood of said patient to determine how many CMMC are in the sample, wherein the processing comprises the method of any of claims 1-8; and (b) determine by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range. The method of claim 9 wherein the normal range of CMMC in a patient sample is less than 7 CMMC in about 2 ml to 10 ml of blood. The method of claim 9 further comprising recommending a therapeutic intervention if the number of CMMC is greater than 8 in from about 2 ml to about 10 ml of blood. 12. A method to determine whether a patient undergoing therapeutic intervention is reducing the number of CMMC or to determine whether a patient who had an abnormal plasma cell disease and who has been successfully treated for such disease remains in remission ; the method comprising (a) processing the blood of said patient to determine how many CMMC are in the sample at a first point in time, wherein the processing comprises the method of any of claims 18; (b) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range; (c) processing the blood of said patient to determine how many CMMC are in the sample at a second point in time, wherein the processing comprises the method of any of claims 18; and (d) determining by counting whether the number of CMMC present in said sample is equal to or greater than or equal to the normal range; (e) compare the numbers in steps (b) and (d). 13. A reagent for capturing circulating multiple myeloma cells comprising colloidal magnetic particles and at least two ligands, wherein the at least two ligands comprise anti-CD138 and anti-CD38.